The long-term goal of my laboratory is to formulate an understanding of the molecular mechanisms operating to regulate expression of class II genes. To this end, the overall aim of this proposal is to continue and expand studies now in progress on the mechanisms by which genes are maintained in a repressed state. This will be accomplished through the characterization of the proteins involved, and their various protein-DNA and protein-protein interactions. Studies on specific transcription of class II genes have indicated that most of the complexity and regulation of RNA synthesis catalyzed by RNA polymerase H probably resides in the initiation step - the recognition of promoter sequences. Proteins have been isolated that together can reconstitute specific transcription from many class I promoters. Much of the emphasis has been on the isolation of regulatory factors, such as activators. coactivators, repressors and corepressors. In most instances, the genes encoding these particular regulators, as well as all of the general transcription factors have been cloned. However, a description of the exact mechanisms controlling gene expression at the transcriptional level hinges on an understanding of this complex process mechanistically using physiological templates. The transcription field has now moved towards reconstituting transcription using chromatin templates. Our studies on promoter recognition by the general transcription factors of the RNA polymerase I transcription system, including repression and anti-repression, have broad implications for the mechanism of RNA synthesis in general and will provide the basis for understanding how specific transcription (actors can modify the transcriptional activity of a particular gene or a set of genes within the context of chromatin and/or using natural promoters. It is likely that these studies will yield basic principles from which the regulatory mechanisms of class II gene expression can be discerned at the molecular level. To this end, the overall aim of this proposal is to continue and expand studies on the mechanisms by which a specific repressor complex, NC2, composed of two subunits Dri and Drapi, operates to regulate gene expression. I propose studies using three different approaches. One approach is directed towards understanding the biochemical properties of the factor. The second approach is directed towards developing an understanding of the role of Dr1 and Drap1 in vivo, using a simple organism, yeast, with which we can apply genetics and study the relationships between Dr1 and Drap1 and other transcription factors. The third approach is directed towards formulating an understanding of the role of the factor in a multicellular organism and thus I describe studies in the mouse.